Mohd Imran Shah, K. S. C. Bose, S. Meenakshisundaram
{"title":"Cloning, Expression and Characterisation of Newly Mined Ketoreductase in Komagataella phaffii","authors":"Mohd Imran Shah, K. S. C. Bose, S. Meenakshisundaram","doi":"10.1134/S000368382560037X","DOIUrl":null,"url":null,"abstract":"<p>Optically active alcohol synthesis has been widely investigated, as they represent an important category of precursors for fine chemicals and pharmaceuticals. This study focused on the expression, purification, and characterization of the gene encoding 2,4-dienoyl-CoA reductase mined from the genome of <i>Komagataella phaffii</i>, here used as a ketoreductase. The impact of the promoter on the expression level of the secreted ketoreductase (<i>kp</i>KRED) was investigated. The <i>kp</i>KRED gene was inserted into an expression vector that had either an inducible alcohol oxidase 1 promoter or a constitutive glyceraldehyde-3-phosphate dehydrogenase promoter. The expressed <i>kp</i>KRED was purified by anion exchange chromatography and the specific activity was found to be 2902 U/mg. A catalytic efficiency <i>K</i><sub>cat</sub>/<i>K</i><sub>M</sub> of 3.80 was observed for ethyl acetoacetate, with <i>K</i><sub>M</sub> value of 4.68 for p-methoxy acetophenone. The purified <i>kp</i>KRED reduced the ketones with optimal activity at temperature 35°C and pH 6.5. Moreover, pure <i>kp</i>KRED exhibited broad substrate specificity towards ketones and ketoesters, with the maximum activity observed for ethyl acetoacetate. It was found that <i>kp</i>KRED, is capable of utilizing both NADH and NADPH as cofactors. In addition, purified <i>kp</i>KRED exhibits efficient solvent tolerance towards 5 organic solvents, such as ethyl alcohol and iso-propylalcohol. This enzyme retained catalytic activity in 10% organic solvents, indicating its strong resilience to organic solvents highlighting its robustness and versatility. This extensive investigation emphasizes the capability of <i>kp</i>KRED as a reliable and adaptable biocatalyst for industrial applications.</p>","PeriodicalId":466,"journal":{"name":"Applied Biochemistry and Microbiology","volume":"61 5","pages":"844 - 855"},"PeriodicalIF":1.1000,"publicationDate":"2025-08-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Applied Biochemistry and Microbiology","FirstCategoryId":"99","ListUrlMain":"https://link.springer.com/article/10.1134/S000368382560037X","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q4","JCRName":"BIOTECHNOLOGY & APPLIED MICROBIOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
Optically active alcohol synthesis has been widely investigated, as they represent an important category of precursors for fine chemicals and pharmaceuticals. This study focused on the expression, purification, and characterization of the gene encoding 2,4-dienoyl-CoA reductase mined from the genome of Komagataella phaffii, here used as a ketoreductase. The impact of the promoter on the expression level of the secreted ketoreductase (kpKRED) was investigated. The kpKRED gene was inserted into an expression vector that had either an inducible alcohol oxidase 1 promoter or a constitutive glyceraldehyde-3-phosphate dehydrogenase promoter. The expressed kpKRED was purified by anion exchange chromatography and the specific activity was found to be 2902 U/mg. A catalytic efficiency Kcat/KM of 3.80 was observed for ethyl acetoacetate, with KM value of 4.68 for p-methoxy acetophenone. The purified kpKRED reduced the ketones with optimal activity at temperature 35°C and pH 6.5. Moreover, pure kpKRED exhibited broad substrate specificity towards ketones and ketoesters, with the maximum activity observed for ethyl acetoacetate. It was found that kpKRED, is capable of utilizing both NADH and NADPH as cofactors. In addition, purified kpKRED exhibits efficient solvent tolerance towards 5 organic solvents, such as ethyl alcohol and iso-propylalcohol. This enzyme retained catalytic activity in 10% organic solvents, indicating its strong resilience to organic solvents highlighting its robustness and versatility. This extensive investigation emphasizes the capability of kpKRED as a reliable and adaptable biocatalyst for industrial applications.
期刊介绍：
Applied Biochemistry and Microbiology is an international peer reviewed journal that publishes original articles on biochemistry and microbiology that have or may have practical applications. The studies include: enzymes and mechanisms of enzymatic reactions, biosynthesis of low and high molecular physiologically active compounds; the studies of their structure and properties; biogenesis and pathways of their regulation; metabolism of producers of biologically active compounds, biocatalysis in organic synthesis, applied genetics of microorganisms, applied enzymology; protein and metabolic engineering, biochemical bases of phytoimmunity, applied aspects of biochemical and immunochemical analysis; biodegradation of xenobiotics; biosensors; biomedical research (without clinical studies). Along with experimental works, the journal publishes descriptions of novel research techniques and reviews on selected topics.
求助内容：
应助结果提醒方式：
京公网安备 11010802042870号
